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Relationship between disease activity indices and colonoscopic findings in patients with colonic inflammatory bowel disease.

The Crohns disease activity index, a similar index devised for patients with ulcerative colitis, and other commonly used laboratory indicators of disease activity have been studied in 50 patients with colonic inflammatory bowel disease undergoing routine colonoscopic assessment and compared with the histological extent and activity of disease. There was only poor correlation between the colonoscopic or histological findings and the indices of disease activity studied, showing that these are not reliable measures of disease activity or extent at the tissue level.

Transcriptome Profile of Human Colorectal Adenomas

Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the β-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation. (Mol Cancer Res 2007;5(12):1263–75)

Effects of Different Doses of Fish Oil on Rectal Cell Proliferation in Patients With Sporadic Colonic Adenomas

BACKGROUND/AIMS Fish oil supplementation can reduce cytokinetic anomalies in the flat rectal mucosa of patients with sporadic colorectal adenoma. This study attempted to identify an optimum dose for fish oil supplementation and evaluate the persistence of its effects during long-term administration. METHODS In a double-blind study, 60 patients with sporadic adenomas received 2.5, 5.1, or 7.7 g of fish oil per day or placebo for 30 days. [3H]thymidine autoradiographic labeling indices were calculated in flat rectal mucosal biopsy specimens collected before and after supplementation. In a subsequent study, 15 patients with polyps received 2.5 g of fish oil per day. Proliferative parameters, mucosal fatty acids, and mucosal and plasma alpha-tocopherol levels were evaluated before, during, and after 6 months of supplementation. RESULTS Mean proliferative indices and mucosal arachidonic acid levels decreased significantly (and to similar degrees) in all treated groups, whereas mucosal eicosapentaenoic and docosahexaenoic acid levels increased. Significantly reduced proliferation was observed only in patients with abnormal baseline patterns. These effects persisted during long-term, low-dose treatment. A transient reduction in mucosal (but not plasma) alpha-tocopherol levels was observed after 1 month of treatment. Side effects were insignificant. CONCLUSIONS Low-dose fish oil supplementation has short-term and long-term normalizing effects on the abnormal rectal proliferation patterns associated with increased colon cancer risk.

The Stool Antigen Test for Detection of Helicobacter pylori after Eradication Therapy

Context Standard treatment regimens do not eradicate infection in approximately 10% to 20% of people with ulcers or gastritis caused by Helicobacter pylori. Symptoms do not reliably identify patients who have persistent infection despite treatment. Although positive results on a urea breath test done 4 weeks after treatment reliably identify persistent infection, a noninvasive test that detects successful eradication earlier would be useful. Contribution This multicenter study shows that a positive finding on a stool antigen test done as early as 1 week after treatment identifies about 95% (range, 70% to 100%) of cases of persistent infection. Generalization Cautions Findings are from patients with dyspepsia who were referred for endoscopy; 20% of patients were still infected at 1 month despite eradication therapy. The Editors Noninvasive tests for Helicobacter pylori are important in primary care, both for initial diagnosis of H. pylori infection and for confirmation of eradication. Current guidelines recommend noninvasive testing and treatment of young dyspeptic patients without alarm symptoms (such as dysphagia or weight loss that suggest underlying malignant disease) in a primary care setting by using low-cost noninvasive tests (1, 2). Randomized, controlled trials have shown that a test and eradicate strategy toward H. pylori is effective in patients with dyspepsia seen in primary care settings who have not undergone investigations such as endoscopy or radiographic studies (3). Post-therapy testing is also growing in importance. Resistant strains of H. pylori are now widely prevalent in the United States and Europe, and eradication therapy with current regimens fails in 10% to 20% of patients (4, 5). Furthermore, some patients with ulcer disease remain symptomatic despite successful eradication of H. pylori and healing of the ulcer (6). In patients with persistent symptoms, testing for persistent H. pylori infection is important to direct further therapy. Routine testing to confirm eradication in patients with complicated ulcer disease, such as bleeding peptic ulcer, is necessary because the risk for rebleeding is greatly increased in patients with persistent infection (7). The choice of tests in the post-therapy setting is limited. Serologic tests are unreliable in determining eradication (8). Endoscopic tests (rapid urease test, histologic examination, or culture) are reliable, but endoscopy is expensive and inconvenient. Until recently, the only noninvasive test that reliably demonstrated whether eradication was successful was the urea breath test (9). This test has high sensitivity and specificity in the post-therapy setting but cannot be used until 4 weeks after treatment. Moreover, the breath test is still not widely available in the United States. The fecal antigen test is a relatively new noninvasive test for detection of H. pylori (10). This test detects the presence of infection by measuring the fecal excretion of H. pylori antigens. It has been approved by the U.S. Food and Drug Administration for detection of H. pylori before and after therapy. We sought to determine whether a stool antigen test administered at various times after treatment correctly identifies persons in whom H. pylori infection persists despite eradication therapy. Methods We prospectively studied 84 patients infected with H. pylori at six clinical centers (31 in Bologna, Italy; 29 in Amsterdam, the Netherlands; 9 in Rome, Italy; 8 in Lisbon, Portugal; 4 in Madrid, Spain; and 3 in Milwaukee, Wisconsin). The sample consisted of consecutive patients with dyspepsia (defined as pain or discomfort centered in the upper abdomen) who were referred by primary care physicians for upper endoscopy (11). Consenting patients were enrolled if they tested positive for H. pylori on endoscopic tests. Patients enrolled in this study have not been enrolled in other studies. Patients were excluded if they had taken proton-pump inhibitors, H2-receptor antagonists, nonsteroidal anti-inflammatory agents, or antibiotics in the 4 weeks before the study. Failure to return for follow-up endoscopy was an a priori exclusion criterion. All patients gave written informed consent, and the study was approved by the human subjects review committee or equivalent at each participating institution. At baseline, patients underwent endoscopy with biopsy sampling for histologic examination (two samples from the antrum and two from the corpus), culture (two samples from the antrum and two from the corpus), and a rapid urease test (one sample from the antrum). All patients were infected with H. pylori at baseline, as demonstrated by positive results on both rapid urease testing and histologic examination or a positive culture for H. pylori. Within 24 hours of the endoscopy, all patients underwent a 13C or 14C urea breath test. The breath test was chosen according to local availability and experience, but in all cases a validated breath test analysis system was used. Cut-off values were determined according to the recommendations of the various manufacturers of these tests. Patients collected stool using a kit consisting of a plastic spoon that is used to scoop a small amount of stool from the toilet paper or toilet bowl into an airtight container. At all sites, the stool assay was performed by using the Premier Platinum HpSA test (Meridian Diagnostics, Inc., Cincinnati, Ohio). The assay is a microwell-based enzyme immunoassay that uses polyclonal antiH. pylori capture antibody adsorbed to microwells. Diluted patient samples and a peroxidase-conjugated polyclonal antibody were added to the wells and incubated at room temperature for 1 hour. A wash was performed to remove unbound material. Substrate was added and incubated for 10 minutes at room temperature. Color develops in the presence of bound enzyme. Stop solution was added, and the results were inspected spectrophotometrically at 450 nm within 15 minutes of adding the stop solution. Visual determination can also be used; this has been shown to have similar results (12). A positive control and a negative control are built into the test. The cut-off values were classified as negative (<0.140), indeterminate (0.140 to 0.159), or positive (>0.160). After completion of the baseline procedures, treatment was begun with ranitidine bismuth citrate (400 mg twice daily) or omeprazole (20 mg twice daily) in combination with amoxicillin (1 g twice daily) and clarithromycin (500 mg twice daily) for 7 to 10 days. Seven-day eradication therapy was used in Europe, where it is approved by the European Union and has been shown to be effective (5). Ten-day triple therapy with proton-pump inhibitors was used in the United States, where it is approved by the U.S. Food and Drug Administration. Patients collected stool for the stool antigen test on days 3, 7, 15, 21, 28, and 35 after completion of H. pylori eradication therapy. On day 35 after completion of eradication therapy, endoscopy was repeated and biopsy samples were again obtained for histologic examination, culture, and the rapid urease test, as performed at the baseline visit. The 13C or 14C urea breath test was repeated on day 35 by using the same method and cut-off values as at baseline. Patients were classified as being infected with H. pylori at baseline and having persistent infection on day 35 if culture of gastric biopsy specimens was positive for H. pylori or results of the rapid urease test and histologic examination were positive. All other patients were classified as negative. These criteria have been recommended by an expert panel for use in clinical trials of H. pylori eradication (13). At baseline, the sensitivity of the stool test and urea breath test were calculated by using the presence of infection (defined above) as the gold standard. At each time point after completion of therapy (days 3, 7, 15, 21, 28), predictive values were calculated by using continued infection on day 35 as the gold standard (positive result on culture or on rapid urease test and histologic examination). Trained investigators who were blinded to the results of the other diagnostic studies performed the stool assays. The first endoscopy procedure was performed before the stool and breath tests. Therapy was given on the basis of results on endoscopic testing. Endoscopists were blinded to the results of post-treatment stool studies and the breath test until all evaluations were completed. Long-Term Follow-up Patients in whom eradication of H. pylori was successful were eligible for entry into a long-term study evaluating the stool antigen test. For 6 months, stool antigen tests were done monthly and a urea breath test was obtained every 3 months. Statistical Analysis Statistical analysis was performed by using StatView for Windows, version 5.01 (SAS Institute, Inc., Cary, North Carolina). Results are presented as the mean (SD). Sensitivity, specificity, probabilities, and predictive values are presented with 95% exact binomial CIs. Equivocal stool tests are considered by inclusion in the denominator of sensitivity and specificity. Stool antigen concentrations at individual time points were compared by using theMannWhitney test with downward adjustment of the P values for repeated observations (14). Role of the Funding Source The manufacturer (Meridian Diagnostics, Inc.) provided the stool kits. The study had no other funding source. Collection, analysis, and interpretation of the data, including the decision to publish, were solely the decision of the authors; the manufacturer of the test had no role in this process. Results The mean age of the 84 study patients was 52 years (range, 18 to 81 years). Fifty-three patients were women, and 31 were men. Endoscopic findings were as follows: normal (7 patients), esophagitis (2 patients), erythema in the antrum (45 patients), erosions in the antrum (11 patients), erosive duodenitis (9 patients), duodenal ulcers (8 patients), gastric ulcer (2 p

Hereditary nonpolyposis colorectal cancer: Review of clinical, molecular genetics, and counseling aspects

Lynch syndrome, or hereditary nonpolyposis colon cancer (HNPCC), is an autosomal-dominant disease accounting for approximately 1-5% of all colorectal cancer cases. Due to the lack of pathognomonic morphological or biomolecular markers, HNPCC has traditionally posed unique problems to clinicians and geneticists alike, both in terms of diagnosis and clinical management. Recently, novel insight into the pathogenesis of this syndrome has been provided by the identification of its molecular basis. In HNPCC families, germline mutations in any of four genes encoding proteins of a specialized DNA repair system, the mismatch repair, predispose to cancer development. Mutations in mismatch repair genes lead to an overall increase of the mutation rate and are associated with a phenotype of length instability of microsatellite loci. The present report summarizes the clinicopathological aspects of HNPCC and reviews the most recent molecular and biochemical findings.

Characterization of MSH2 and MLH1 mutations in Italian families with hereditary nonpolyposis colorectal cancer

Mismatch repair genes MSH2 and MLH1 are considered to be the two major genes that are responsible for hereditary nonpolyposis colorectal cancer (HNPCC). Germline heterozygous inactivating mutations of MSH2 and MLH1 have been identified previously in a substantial fraction of individuals who are predisposed genetically to colorectal carcinoma (CRC) and other tumors of the HNPCC spectrum. With the aim of determining the relevance of these two genes in the Italian population, we submitted to mutational analysis a set of 17 HNPCC families, all of which fulfilled the “Amsterdam criteria.” A combination of different techniques, including reverse transcription‐polymerase chain reaction (RT‐PCR) of long fragments and single‐strand conformation polymorphism (SSCP) on cDNA and genomic DNA, allowed the identification of ten molecular variants, seven of which are predicted to inactivate mismatch repair function. The mutated predisposing gene was MSH2 in two families and MLH1 in five other families. All of the mutations were characterized by DNA sequencing and appeared to involve different molecular mechanisms, such as short in‐frame and out‐of‐frame deletions, splicing errors, and nonsense mutations. This study also demonstrates that, in the Italian population, a considerable fraction of HNPCC families (at least 41%) is linked to MSH2 and MLH1 mutations. Genes Chromosom. Cancer 18:8–18, 1997.

Severe imbalance of cell proliferation and apoptosis in the left colon and in the rectosigmoid tract in subjects with a history of large adenomas

BACKGROUND Alterations in epithelial proliferation and apoptosis in colonic mucosa are associated with an increased risk of colon cancer. It is unclear if these alterations represent a generalised “field defect”. AIMS To analyse segmental patterns of cell proliferation and apoptosis in the colon of subjects with a high and no apparent risk of colon cancer. METHODS Pancolonoscopy was performed in 15 patients with resected adenomas (⩾1.5 cm) and in nine subjects without an apparent risk of colorectal cancer. Mucosal biopsies were taken from the right colon, left colon, and sigmoid rectum. Crypt cell proliferation and apoptosis were evaluated, respectively, with bromodeoxyuridine immunohistochemistry and terminal deoxyuridine nucleotidyl nick end labelling of DNA strand breaks. Results are expressed as total labelling index (TLI) and labelling index (LI) for each of the five compartments in which colonic crypts were divided (fourth and fifth compartments were evaluated together) for cell proliferation and as apoptotic index (AI) for apoptosis assessment. RESULTS No significant segmental variations in proliferation were found in either group. Compared with controls, adenoma patients had higher TLIs for the right (p>0.05), left (p<0.005), and sigmoid rectum (p<0.05) segments, and higher left colon LIs for crypt compartments (compartment 1, p<0.01; compartment 2, p<0.005; compartment 3, p<0.001; compartments 4–5, p<0.01). Control AIs were similar in all segments but in the adenoma patients left colon and sigmoid rectum AIs were lower than their right colon indexes (p<0.05, p<0.05) and corresponding values for controls (p<0.01, p<0.05). CONCLUSIONS The colonic mucosa of patients with past adenomas presents diffuse hyperproliferation and, distally, abnormally distributed proliferating cells and markedly reduced apoptosis. These changes represent a significant risk for malignancies and could account for the high prevalence of left colon tumours.

Importance of changes in epithelial cell turnover during Helicobacter pylori infection in gastric carcinogenesis.

The role of Helicobacter pylori in gastric carcinogenesis is supported almost exclusively by epidemiological data and prospective histopathological studies. From biological and molecular points of view, there is no evidence that H pylori or its cytotoxic products have any mutagenic effects. Nevertheless, this infection is associated with profound changes in the pattern of epithelial cell turnover in gastric glands, though the importance of these changes in gastric carcinogenesis is still controversial. H pylori infection increases cell proliferation and alters the distribution of cycling cells within these glands, but these changes can be reversed by successful eradication of the infection. Apoptosis seems to be increased in gastric epithelial cells during H pylori infection, as shown by in vitro studies. There is some, though no conclusive, evidence that this finding also occurs in H pylori positive subjects. It seems that cagA status influences the effect of H pylori on epithelial apoptosis in infected patients. An association of in vitro H pylori induced apoptosis with changes in the expression of pro- and anti-apoptotic genes is reported in the literature, but further study is necessary to clarify the effect of H pylori infection on the molecular events of the apoptotic pathway.

Rectal epithelial cell proliferation patterns as predictors of adenomatous colorectal polyp recurrence.

To determine whether proliferative patterns in flat rectal mucosal samples can predict the recurrence of adenomatous colorectal polyps, after polypectomy, biopsy specimens from normal looking rectal mucosa were obtained at endoscopy from 55 patients diagnosed for the first time as having adenomatous colorectal polyps. Epithelial cell proliferation was assessed in biopsy specimens through 3H-thymidine autoradiography. After polypectomy, patients were followed for 24 months and underwent complete colonoscopy every 6 months to detect and remove any metachronous lesions. In 40 patients second biopsy specimens were taken during one of the follow up colonoscopies to evaluate the stability of proliferative indices over time. The ratio of labelled (S phase) to total cells (labelling index) for the entire crypt, as well as ratios for each of the five equal compartments into which the crypt had been divided longitudinally, was calculated for each patient. Mean labelling indices for upper crypt compartments 3 and 4 + 5 in the 22 patients in whom polyps recurred were significantly higher (respectively p < 0.05 and p < 0.01) than those of the 33 without recurrence suggesting that an upward shift of the crypts replicative compartment is associated with polyp recurrence. Labelling indices remained essentially unchanged in those patients who underwent biopsy twice. Reproducible kinetic parameters such as these might be useful in planning follow up of patients with adenomatous polyps after polypectomy.

Genetic testing among high-risk individuals in families with hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is frequently associated with constitutional mutations in a class of genes involved in DNA mismatch repair. We identified 32 kindreds, with germline mutations in one of three genes hMSH2, hMLH1 or hMSH6. In this study, we purposed to evaluate how many high-risk individuals in each family underwent genetic testing: moreover, we assessed how many mutation-positive unaffected individuals accepted colonoscopic surveillance and the main findings of the recommended follow-up. Families were identified through a population-based registry, or referred from other centres. Members of the families were invited for an education session with two members of the staff. When a kindred was consistent with HNPCC, neoplastic tissues were examined for microsatellite instability (MSI) and immunohistochemical expression of MSH2, MLH1 and MSH6 proteins. Moreover, constitutional mutations were searched by SSCP or direct sequencing of the whole genomic region. Of the 164 subjects assessed by genetic testing, 89 were gene carriers (66 affected – that is, with HNPCC-related cancer diagnosis – and 23 unaffected) and 75 tested negative. Among the 23 unaffected gene carriers, 18 (78.3%) underwent colonoscopy and four declined. On a total of 292 first degree at risk of cancer, 194 (66.4%) did not undergo genetic testing. The main reasons for this were: (a) difficulty to reach family members at risk, (b) lack of collaboration, (c) lack of interest in preventive medicine or ‘fatalistic’ attitude towards cancer occurrence. The number of colorectal lesions detected at endoscopy in gene carriers was significantly (P<0.01) higher than in controls (noncarriers). We conclude that a large fraction of high-risk individuals in mutation-positive HNPCC families does not undergo genetic testing, despite the benefits of molecular screening and endoscopic surveillance. This clearly indicates that there are still barriers to genetic testing in HNPCC, and that we are unable to provide adequate protection against cancer development in these families.